A. Field Of The Invention
The present invention is related generally to the detection of protein; and more particularly, to a novel class of protein error indicators.
B. Description Of The Background Art
The detection of protein is important in the diagnosis of disease, in medical research and in industry. Several methods exist for the detection of protein in a sample. With the exception of the methodologies using protein error indicators, all of the protein detection techniques are multi-step processes, often requiring highly complex equipment and several hours to complete.
One technique commonly used to measure protein in a sample is the Biuret method. According to this method, the sample is first acidified to precipitate any protein in the sample. The precipitated protein is then re-solubilized in a moderately alkaline medium and treated with a solution containing cupric ions. The peptide bonds of the protein and the cupric ion react to form a colored chelate. The absorbance of the treated solution is then determined using a spectrophotometer. From this data, the amount of protein in the sample is estimated using calibrated spectrophotometric absorbance curves. This method generally takes form 1 to 3 hours to perform.
A variation of the Biuret method is the Lowry method. According to the Lowry method, after the precipitated protein is re-solubilized, a phosphotungstomolybdic acid reagent is added to the solution under alkaline conditions to oxidize any phenolic compounds in the solution. Inasmuch as substantially all proteins contain some phenolic compounds, e.g., tyrosine, this technique is capable of measuring protein in a sample. The absorbance of the treated solution is then measured with a spectrophotometer. Using calibrated spectrophotometer curves, the measured absorbance is thereafter used to estimate the amount of protein in the sample. A number of buffers and other compounds, however, containing amine groups, e.g., TRIS, glycine and amide buffers, interfere with the test.
Another method used to determine the presence of protein in a sample is measuring turbidity following sample acidification. According to the method, the turbidity of the sample is measured using a spectrophotometer following the addition of a protein precipitating agent, generally an acidifying agent, to the sample. The calculated turbidity of the sample is compared to spectrophotometeric standard curves to determine the presence of protein in the sample. Common precipitating agents used in this method include sulfosalicylic acid, trichloracetic acid and tannic acid.
Methodologies using protein error indicators are widely used to determine the presence of protein in a sample. Methods using protein error indicators are inexpensive, fast, simple and convenient. Phenolsulfonephthalein compounds, such as bromophenol blue, bromocresol green and coosmassie blue, are, perhaps, the most widely used protein error indicators. Methodologies using protein error indicators often involve reagent strips which are impregnated with the protein error indicator. According to these methods, the reagent test strip is contacted with a small quantity of the sample. If protein is present in the sample, the test strip will indicate this by simply changing color. The color observed may vary depending on the concentration of protein in the sample. This variable color change is used to quantify the protein in the sample. Reagent strips of the above-type require a minimum of training to use correctly. These reagent test strips provide an accurate, convenient, and rapid vehicle for the on-the-spot determination of protein. Test papers such as these are widely used by technicians in industry, research and clinical laboratories.
In more detail, protein error indicators are pH indicators including an ionizable group which has a pKa value that is displaced by the presence of protein. In the case of the phenolsulfonephthaleins, the ionizable group is a phenolic hydroxyl. The release of the proton from the phenolic hydroxyl causes the observable color change which is indicative of protein in the sample being tested. Protein error indicators which are generally considered useful for the analytical determination of protein in a sample are described in U.S. Pat. No. 4,013,416.